Tissue repair compositions and methods for their manufacture and use

ABSTRACT

An osteogenic composition is prepared by a process including the steps of subjecting demineralized bone to an extraction medium to produce an insoluble extraction product and a soluble extraction product, separating the insoluble extraction product and the soluble extraction product, drying the soluble extraction product to remove all or substantially all of the moisture in the soluble extraction product, and combining the dried soluble extraction product of step c) with demineralized bone particles. Preferably, the process does not involve heating.

This application claims priority to U.S. Provisional Patent ApplicationSer. No. 60/316,005, filed Aug. 30, 2001, the entire contents of whichis incorporated herein by reference.

This invention relates to tissue repair compositions and methods formanufacturing and using such compositions.

BACKGROUND OF THE INVENTION

Various compositions have been used to repair damaged tissues.Compositions are available to provide a scaffold to support new bonegrowth and/or to provide factors that induce new bone growth.Demineralized bone particles (also referred to as demineralized bonematrix or DBM) and bone morphogenetic proteins (BMPs) are two materialsthat have been used to enhance bone growth. For example, Jefferies (U.S.Pat. No. 4,394,370) discloses tissue repair compositions containing DBM,BMPs, or both in a reconstituted collagen matrix. Glowacki et al. (U.S.Pat. No. 4,440,750) discloses aqueous compositions of DBM andreconstituted collagen fibers.

DBM is generally composed of particles of bone tissue that have beenspecially treated, generally by soaking in acid, to remove their mineralcontent. The resulting DBM is composed mainly of highly cross-linkedcollagen. The remaining non-collagenous proteins include proteins suchas TGF-β, PDGF, osteopontin, osteonectin, BMPs, and others. BMPs are agroup of proteins categorized in the transforming growth factor betasuper-family of proteins. To date, several BMPs have been isolated andassociated with the bone healing process. BMPs can be isolated from boneas a mixture of proteins or produced individually through recombinantgene technology.

DBM may be used directly in bone repair compositions. See, e.g.,Jefferies, supra; Glowacki et al., supra. However, in such compositions,the tissue repair factors are trapped within the highly cross-linkedcollagen network of the DBM. It is believed that the BMPs and otherembedded tissue repair factors are slowly released as the collagencomponent of DBM is degraded. Therefore, the potential effectiveness ofthe tissue repair factors within the DBM is hindered. An alternative toslow release is to isolate the tissue repair factors from the DBM.Isolated and purified connective tissue repair factors have been used inbone repair compositions, but extraction, purification, and mixture witha dispersion medium or incorporation into a delivery vehicle requiresmultiple steps.

There is a need in the art for additional tissue repair compositionsthat employ tissue repair factors that are substantially freed of thecross-linked DBM collagen network and that do not require complicatedextraction and purification steps.

SUMMARY OF THE INVENTION

This invention relates to tissue repair compositions comprising solubleand/or insoluble products from the extraction of DBM, and methods fortheir manufacture and use. These DBM-derived products, also referred toherein as the “soluble extraction product” and the “insoluble extractionproduct” may contain tissue repair factors and can be processed toproduce a variety of formulations and consistencies.

The compositions according to the invention use the soluble and/orinsoluble products from the extraction of DBM. The DBM extraction isgenerally conducted at room temperature in a suitable extraction medium.Following extraction, the soluble and insoluble extraction products areseparated. These products may be further processed, for example, bycentrifuging, decanting, filtering, titration, precipitation, dialyzing,fully or partially drying, rehydrating and sterilizing. In a preferredembodiment, these steps are performed without heating. These productsmay be used in a variety of connective tissue repair compositions, aloneor in combination with other active or inactive ingredients.

In a preferred embodiment, the inventive compositions contain arehydrated form of the dried soluble extraction product. The rehydratedsoluble extraction product can be used alone or in a mixture with one ormore active or nonactive ingredients. For example, the rehydratedsoluble extraction product can be combined with one or more biologicallyactive materials and a thickening agent. The physical properties of theresulting mixture(s), including viscosity, can be varied by modifyingthe relative concentrations of the soluble extraction product, the sizeof the pieces of dried soluble extraction product, the amount of waterused for rehydration, the extent of any subsequent drying, and othersoluble or insoluble ingredients. For example, the final composition mayhave the consistency of a gel, paste, putty or sponge.

The compositions according to the invention can be prepared forinjection or insertion at, into, onto, or near bone defect sites,cartilage repair sites, or other musculoskeletal sites. The inventivecompositions can also be used as a coating on surgical implants to beinserted at, into, onto, or near bone defect sites, cartilage repairsites, or other musculoskeletal sites. Accordingly, the invention isdirected to an osteogenic surgical implant comprising surgical implantcoated with the inventive osteogenic compositions and a method oftreating a bone defect comprising providing a surgical implant, coatingthe surgical implant with the inventive osteogenic composition, andimplanting the surgical implant at a bone defect site.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a color photograph of the composition from Example 1 plusresidual putty implanted for 28 days in an athymic mouse. The photographshows a bone ossicle (arrow) surrounding fatty bone marrow (BM).Residual material (R) is still present in this explant. Haematoxylin andeosin stain; original magnification ×200.

FIG. 1B is a color photograph that shows the same sample at a differentsite. Additional new bone (arrows) and bone marrow (B) is apparent.Residual material (R) is also present in this site. Haematoxylin andeosin stain; original magnification ×200.

FIG. 2A is a color photograph of the composition of Example 2 plus DBMputty implanted for 28 days in an athymic mouse. The photograph showsmultiple foci of new bone (arrows) and bone ossicles surrounding fattybone marrow. Residual DBM is still present in this explant. Haematoxylinand eosin stain; original magnification ×100.

FIG. 2B is a color photograph at higher magnification, wherein the wovennature of the new bone of one of the ossicles seen in FIG. 2A isapparent. The woven bone surrounds an area of healthy bone marrow (BM).Haematoxylin and eosin stain; original magnification ×400.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

An osteogenic composition is prepared by a process including the stepsof subjecting demineralized bone (DBM) to an extraction medium toproduce an insoluble extraction product and a soluble extractionproduct, separating the insoluble extraction product and the solubleextraction product, drying the soluble extraction product to remove allor substantially all of the moisture in the soluble extraction product,and combining the dried soluble extraction product of step c) withdemineralized bone particles. Preferably, the process does not involveheating.

The DBM used in the extraction process according to the invention can beprepared according to a variety of different methods. Conventionalmethods, such as those identified in Jefferies, supra, and Glowacki etal., supra are preferred. Such conventional methods for preparing DBMinclude a defatting step and a demineralization step. Different methodsof defatting, e.g., hot water, or chloroform/methanol washes, can beused. Demineralization can be performed according to a variety ofdifferent methods, generally using different types of acid solutions forvarying times and at variable temperatures, to remove all orsubstantially all of the mineral content from the bone.

For purposes of this invention, any shape and particle size of DBM maybe used. This includes DBM in the form of fragments, slices, pellets,shavings, strips, granules, or powder as well as demineralized wholebones. Preferably, the demineralized bone is of small particle size, andmost preferably in the form of granules or powder. Most preferably, thedemineralized bone is in the form of particles having an averageparticle size of from about 100 to about 1000 microns, furtherpreferable from about 125 to about 850 microns.

In the method for manufacturing the compositions according to theinvention, DBM is placed in an aqueous-based medium capable ofextracting collagen, gelatin, and/or connective tissue factors. Thisstep is performed by a method that is different than conventionalmethods used to extract gelatin (LaRoche, et al. (U.S. Pat. No.5,908,921), Lilja, et al. (U.S. Pat. No. 5,877,287), and Rainville, etal. (U.S. Pat. No. 6,080,843)), because it is performed at roomtemperatures and uses agitation unlike other described methods (e.g., asdescribed in O'Leary, et al. (U.S. Pat. No. 5,236,456)). The extractioncauses limited hydrolysis of chemical bonds within the collagen (MillerE. J. & Gay S., Collagen: An Overview, In: Methods in Enzymology, vol.82 (A), pp. 3–32, 1982). This results in the production of a DBM-derivedprotein mixture previously characterized as water-soluble collagen andits lower molecular weight cleavage products, collectively referred toas “gelatin”. (Rainville et al. supra; Nagumo et al. Kitasato Arch. Exp.Med. 48: 189–191, 1975; Batge et al., Eur. J. Biochem. 192: 153–159,1990). Due to the hydrolytic cleavage of collagen's intra- andintermolecular bonds, the extraction process also releases some of theconnective tissue repair factors that were embedded in the collagenmatrix of the DBM. The released connective tissue repair factors aresoluble in the DBM-derived extract solution.

The extraction may be conducted in medium that is an acid, alkaline orsalt solution. Any of several acids may be used to perform theextraction. Such acids include hydrochloric acid, citric acid, aceticacid, lactic acid, and malic acid. Alternatively, any of severalalkaline solutions (e.g., sodium hydroxide, or potassium hydroxide) oraqueous salts (such as lithium chloride) may be used to perform theextraction. Preferably, a carboxylic acid is used. Most preferably,citric acid is used. The concentration of the acid, base, and/or saltmay vary, depending on the effectiveness of the acid, base, or salt usedas a hydrolytic agent. The amount of DBM placed in the acid, alkaline orsalt solution may vary, depending on the strength of the solution andthe shape and size of the DBM.

Preferably, where citric acid and granular or powdered DBM are used,about 10 to about 50 mL of about 1.0 to 10.0 M citric acid is used pergram of DBM. Most preferably, about 20 mL of about 2.0 to about 3.0 Mcitric acid is used per gram of DBM. The DBM and aqueous acid solutionmay be shaken, stirred, or otherwise agitated to speed this extractionprocess. Preferably the aqueous acid solution and DBM are stirred oragitated between about 2 and about 96 hours, and most preferably,between about 48 and about 72 hours. This extraction step may take placeat any temperature below which endogenous proteins within the DBM beginto irreversibly denature. For the purpose of this invention, it ispreferred that ambient temperature or room temperature is between about15° C. and about 25° C., more preferably between about 18° C. and about25° C., and further preferably between about 15° C. and 21° C.

By varying the parameters of the extraction system, the molecular weightand physical properties of the resulting gelatin can be affected. The pHand/or concentration of the system affect the protein composition of theextraction products. The ability of the system to extract proteins isbased on both the pH and concentration of the DBM and extraction medium.For example, the extraction of collagen and conversion into gelatin viahydrolysis has been shown to be dependent on the pH of the extractionmedia (LaRoche, et al; Lilja, et al.; Rainville, et al., supra).Similarly, the pH and/or concentration of the extraction media affectthe protein composition of the extraction products. The relativeconcentrations of DBM and extraction medium during the extraction stepalso affect the ability and degree to which proteins are extracted.

The extraction step may also be performed under a vacuum. For example,demineralized bone particles and an extraction medium can be contactedin a flask attached to a vacuum line connected to a pump that supplies avacuum, e.g., about 28 mm Hg vacuum.

Following DBM exposure to the extraction medium, the soluble extractionproduct is separated from the insoluble extraction product, which isgenerally in the form of an insoluble solid residue. This separation mayoccur by any of a number of processes, such as decanting, filtering, orcentrifuging. Preferably, filtration is used. Optionally, water may beadded to the remaining insoluble extraction product as a means ofwashing and collecting additional dissolved material. Preferably, thiswater wash step is performed, for example, by adding about 10 to about100 mL of sterile deionized water per gram of DBM starting material,after which the mixture is stirred, shaken, or otherwise agitated thenseparated. Most preferably, about 20 mL of sterile deionized water isadded per gram of DBM starting material, and the mixture is shaken thenfiltered. The liquid phase may be saved and later combined with thesoluble extraction product to increase the product yield. Optionally,the extraction and separation steps may be repeated one or more times byadding fresh extraction medium to the residue followed by an aqueouswash. Following the repeat extractions and water washes, the extractionand water wash volumes may be saved and added to the extraction and/orwater wash volumes from the first extraction to increase the productyield. The insoluble extraction product may be saved and used asdescribed herein.

The soluble extraction product is next diluted, neutralized and/or thesalts are removed. This also may occur by any of a number of processes,such as titration, dialysis, liquid-liquid extraction, or precipitation.Preferably, dialysis is used. A protein concentrator or ultrafiltrationunit may be used before or during dialysis to speed the dilution,neutralization and/or salt removal processes. The neutralization andsalt removal processes should eliminate a substantial portion of thesoluble ions and small molecules in the soluble extraction product.Preferably, if an acid or basic extraction medium is used, the pH of thesoluble extraction product is adjusted to between about 4 and about 10.Most preferably, if an acid extraction medium is used, the pH of theremaining soluble extraction product is adjusted to be between about 0.5and about 5.5, further preferably between about 0.5 and about 3.5.

The neutralized, salt-free soluble extraction product is partially orfully dried to remove excess water. The drying may occur by variousmeans. Preferably, the drying occurs by lyophilization (freeze drying).Preferably, the drying is complete, such that all water is removed and adry product remains. Alternatively, the lyophilization or other dryingprocess may be arrested at some time prior to completion, such that avariety of products exist in concentrated, but not completely dehydratedform. Such products are referred to as “concentrate” herein. The driedsoluble extraction product generally has a white, fluffy appearance likecotton. The concentrate will have a greater moisture content and,therefore, may have the appearance of a gel, putty, or paste.

The dried soluble extraction product or concentrate may be partially orfully rehydrated to a fluid or plastic form, such as a gel, putty, orpaste. The primary components of this material are water, DBM-derivedproteins, and connective tissue proteins. The relative amounts of drysoluble extraction product and water in the mixture may be varied toadjust the viscosity and other physical characteristics of the mixture.Preferably, about 5 to about 15 mL of deionized water is added to about0.1 to about 3.0 g of dry product, and most preferably about 10 mL ofdeionized water is added to about 0.8 g of dry product. The rehydrationprocess may be aided by means of mechanical mixing, such as shaking orstirring at room temperature. Preferably, the rehydration is aided bystirring. Concentrate may be used in place of or in addition to dryproduct. Where concentrate is used, water or dry product may be mixedwith concentrate to change the viscosity and other physicalcharacteristics of the resulting mixture.

One or more biologically active ingredients may be added to theresulting composition. These active ingredients may or may not berelated to the connective tissue repair capabilities of the composition.Suitable active ingredients include DBM and the insoluble extractionproduct containing residual, endogenous bone morphogenetic proteins andrelated proteins such as cartilage derived morphogenetic proteins(CDMPs). Other active ingredients that may be added to the composition,including bone-derived materials such as cortical or cancellous bonechips and bone mineral, osteogenic chemicals (e.g. L-arginine),osteogenic peptides (e.g. OSA), osteogenic growth factors (e.g.transforming growth factor-beta [TGF-β], insulin-like growth factor[IGF], platelet derived growth factor [PDGF], vascular endothelialgrowth factor [VEGF], fibroblast growth factor [FGF]), and recombinantBMPs (e.g. rBMP-2, rBMP-7), fibronectin, and blood-derived proteins.When added in appropriate combinations, these active ingredients mayassist bone repair, cartilage repair, ligament and tendon repair,meniscal repair, and other musculoskeletal applications.

One or more thickening materials may be added to the resultingcomposition. Any such material may also be an active ingredient orbiologically inert. Suitable thickening materials include collagen,insoluble extraction product (which may or may not contain residualBMPs), bone mineral, hydroxyapatite, tricalcium phosphate, biphasiccalcium phosphate, calcium sulfate, biological glasses, and natural orsynthetic polymers. Such polymers include poloxamer 407 and relatedpolymers. Where insoluble extraction product is used as a thickeningmaterial, it is preferably is washed with water to remove any residualextraction medium. Preferably, DBM, insoluble extraction product, and/ora reverse phase medium are used as a thickening material with or withoutadded proteins. The reverse phase medium may be an aqueous mixture ofPluronic F127 (BASF Corp.) in an amount sufficient to confer a reversephase property to the composition, preferably an approximately 20–40%w/w, more preferably about 23–32% w/w and further preferably about 25%w/w or about 35% w/w mixture of Pluronic® F127 and water. Other reversephase media include aqueous mixtures of deriviatives of Pluronic® F127,such as those disclosed in U.S. Provisional Patent Application Ser. No.60/345,113, which is incorporated herein by reference.

The biological, physicochemical and biodegradation properties of thecomposition may be altered by known cross-linking agents such aschemicals (e.g., glutaraldehyde or formaldehyde) or radiation (e.g.gamma or electron beam). Preferably radiation is used as thecross-linking agent, and most preferably electron beam (E-beam)radiation is used to irradiate the wet or dry materials at doses betweenabout 5 and about 50 kGray.

The resulting composition may be used in several different manners. Inone preferred embodiment, the composition is used as a coating forsurgical implants. Preferably, the mixture is applied to lyophilized,cancellous bone chips; or cancellous bone chips are dipped into mixture.The bone chips, coated with the mixture, may be dried. The drying stepmay be conducted by any conventional drying process, includinglyophilization or oven drying. Preferably, drying is by lyophilization.The coated bone chips may be used as or in surgical implants at, in, on,or near bone defect sites, cartilage repair sites, or othermusculoskeletal sites. Alternatively, the coating may be applied tolarger segments of bone, artificial implants, or any other kind ofsurgical implant.

In another preferred embodiment, the composition is injected or insertedat, in, on, or near a bone or chondral defect site. The manner ofinjection or insertion is not essential, but preferably injection is viasyringe and insertion is by creating a surgical opening to access thebone or chondral defect site.

In another preferred embodiment, the composition is mixed with acombination of active and filler or thickening materials such as DBM andinsoluble extraction product respectively, and injected or inserted at,in, on, or near a bone or chondral defect site. Preferably the weight toweight (w/w) ratio of DBM to insoluble extraction product is about 3 to1.

Alternatively, the dry soluble extraction product or concentrate may bemixed with aqueous alcohol or other volatile solutions, cast into adesired shape and dried to form a sponge-like material. Preferably, aone to six carbon alcohol is used. Most preferably, ethanol is used.Preferably, a 1 to 20 percent alcohol by volume solution is used. Mostpreferably, a 4.75 percent ethanol by volume solution is used.Preferably, 20 mg to 200 mg of dry material are combined with each mL ofethanol. Preferably, 50 to 80 mg of dry soluble extraction product permL of ethanol are used. A biologically active ingredient, as discussedabove, may also be added. Preferably, DBM is used. Additionally, one ormore thickening materials, as discussed above, may also be added.Insoluble extraction product may also be added to this composition. Theresulting composition may be cast into a sheet or other shape with orwithout other added materials. The sheet or other shape is dried. Dryingmay be done by any conventional method, including lyophilization orair-drying. Preferably, drying is by lyophilization.

In a preferred embodiment, the sheet or shape formed with an alcoholsolution as described above is used as or as part of a surgical implant.Preferably, where a sheet is used, it is used as a wrap around an areaor as a patch inserted into a bone defect site, e.g., insertion into abone defect, a chondral defect, a spinal fusion cage or a pre-reamedacetabular bed.

In a further preferred embodiment, the dried soluble extraction product,which is in the form of a white, fluffy material is used to make asponge-like material that contains demineralized bone particles. Thewhite, fluffy material may be chopped into small pieces of about 0.5 toabout 5 cm, more preferably about 1 to about 2 cm. The chopped materialis then combined with aqueous ethanol (approximately 3–10% ethanol, morepreferably about 4-5% ethyl alcohol), and mixed until the white, fluffymaterial is dispersed. DBM particles are then added at a ratio of about2–4:1 by weight, more preferably 3:1 by weight and the composition isthoroughly mixed. Then, ethanol is added to the composition and themixed composition is poured into a container, preferably a containerthat is in the shape of the desired product. The composition is thenrefrigerated, frozen and lyophilized to obtain a composition that issubstantially free of moisture. The end product has a sponge-likeconsistency.

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples that follow representtechniques discovered by the inventors to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

EXAMPLES Example 1

2.0 M—2 Day Extraction with Citric Acid

Twenty mL of 2.0 M citric acid is added per gram of DBM in a 500 mLpolypropylene centrifuge tube. The citric acid and DBM are stirred atroom temperature for 48 hours. The DBM/solvent dispersion is filteredthrough a 100 μm mesh. The residual that does not pass through the meshis further washed with water. The wash is filtered and combined with theoriginal filtrate. The filtrate and residual are saved and set aside.

Example 2

3.0 M—2 Day Extraction with Citric Acid

Twenty mL of 3.0 M citric acid is added per gram of DBM in a 500 mLpolypropylene centrifuge tube. The citric acid and DBM are stirred atroom temperature for 48 hours. The DBM/solvent dispersion is filteredthrough a 100 μm mesh. The residual that does not pass through the meshis further washed with water. The wash is filtered and combined with theoriginal filtrate. The filtrate and residual are saved and set aside.

Example 3

3.0 M—3 Day Extraction with Citric Acid

Twenty mL of 3.0 M citric acid is added per gram of DBM in a 500 mLpolypropylene centrifuge tube. The citric acid and DBM are stirred atroom temperature for 72 hours. The DBM/solvent dispersion is filteredthrough a 100 μm mesh. The residual that does not pass through the meshis further washed with water. The wash is filtered and combined with theoriginal filtrate. The filtrate and residual are saved and set aside.

Example 4

3.0 M—5 Day Sequential Extraction with Citric Acid

Twenty mL of 3.0 M citric acid is added per gram of DBM in a 500 mLpolypropylene centrifuge tube. The citric acid and DBM are stirred atroom temperature for 24 hours. The DBM/solvent dispersion is filteredthrough a 100 μm mesh. The filtrate is separated and saved for furtherprocessing. The residual that does not pass through the mesh isrecombined with fresh citric acid (20 ml/g of starting DBM) and stirredfor an additional 24 hours. This process is repeated for five days, suchthat five extractions will have occurred. The filtrate from eachisolation step is kept separate for individual processing. At the fifthday, the residual is rinsed with de-ionized water (20 ml/g of startingDBM) which is combined with the original filtrate. The filtrate andresiduals are saved and set aside.

Example 5

Neutralization and Lyophilization of Supernatant

The filtrate from Example 1, 2, 3, or 4 is placed in dialysis tubing(pore size 8,000–10,000 KDa) and dialyzed against deionized water untilthe pH of the supernatant portions reaches a minimum of 5. At thispoint, the filtrate is transferred to a lyophilization flask, shellfrozen, then placed on a lyophilizer. This lyophilate is referred tobelow as the soluble portion.

Example 6

Processing the Residue

The resulting insoluble particles from Example 1, 2, 3, or 4 are washedwith 200 mL H₂O per gram DBM. This wash is repeated until the particlesreach a pH of 4–8; the wash liquids are discarded. The insolubleparticles are lyophilized to obtain a dry insoluble material.

Example 7

Formulation of an Extrudable Gel

The soluble portion from Example 5 is dissolved in deionized water at aconcentration of 0.08 g soluble portion per ml water. The mixture isstirred at room temperature for approximately 15 minutes to 1 hour oruntil the mixture becomes homogenous. At this point the soluble portionwill have dissolved and a gel begins to form. Gelling may be,accelerated by cooling the suspension/solution to about 1 to 10° C. Thegel is placed at 4° C. for 15 minutes to accelerate gel formation or isleft at room temperature for 1 hour. The resulting gel is stable at roomtemperature. This gel may be osteoinductive at certain concentrationsand used for percutaneous injection or surgical implantation at, in, on,or near bone fracture or defect sites. This gel may be mixed withadditional active or inactive materials.

Example 8

Formulation of an Extrudable Product with Poloxamer 407 as a ThickeningMaterial

Soluble portion from Example 5 is dissolved in deionized water at aconcentration of 0.04 g/ml. The resulting gel (10 ml) is mixed with 20mL of a 35% w/w poloxamer 407 gel. The mixture is stirred at roomtemperature for several minutes or until the mixture becomes homogenousand opaque. This gel may be osteoinductive at certain concentrations andmay be used for percutaneous injection or surgical implantation at bonedefect sites or mixed with additional active or inactive fillers tocreate a putty or paste-like consistency.

Example 9

Formulation of an Extrudable Product with Insoluble Portion as aThickening Material

An extrudable gel, putty or paste is produced by mixing 2.0 ml of thegel from Example 7 with 0.4 g of the insoluble particles from Example 6.The mixture is stirred at room temperature for several minutes until agel, putty, or paste consistency has been obtained. This material may beused for injection or surgical implantation at, in, on, or near boneand/or chondral defect sites.

Example 10

Formulation of an Extrudable Product with DBM as an Active Material

An extrudable gel, putty or paste is produced by mixing 2.0 ml of thegel from Example 7 with 0.6 g of DBM. The mixture is stirred at roomtemperature for several minutes until a gel or putty consistency hasbeen obtained. This material may be used for injection or surgicalimplantation at, in, on, or near bone and/or chondral defect sites.

Example 11

Formulation of an Extrudable Product with DBM and Insoluble Particles asThickening Material

An extrudable gel, putty or paste is produced by mixing 4.76 ml of thegel from example 7 with 1.02 g of DBM and 0.24 g of the insolubleparticles from Example 6. A similar putty with 1.02 g of insolubleresidual and 0.24 g of DBM may also be obtained. The mixture is stirredat room temperature for several minutes until a gel or putty consistencyhas been obtained. This gel may be used for injection at, in, on, ornear bone and/or chondral defect sites.

Example 12

Formulation of an Extrudable Putty with Both Poloxamer 407 and InsolublePortion as Thickening Materials

The gel from Example 8 (3.0 ml) is mixed with 1.5 g of the insolubleparticles from Example 6. The mixture is stirred at room temperature forseveral minutes until a putty consistency has been obtained. Thismaterial may be used for injection or surgical placement at, in, on, ornear bone and/or chondral defect sites.

Example 13

Formulation of an Extrudable Putty with Poloxamer 407, DBM, andInsoluble Portion as Thickening Materials

The gel from Example 8 (3.0 ml) is mixed with 1.2 g of DBM and 0.3 g ofthe insoluble particles from Example 6. A similar putty can be formedfrom 1.2 g of the insoluble particles from Example 6 and 0.3 g of DBM.The mixture is stirred at room temperature for several minutes until aputty consistency has been obtained. This material may be used forplacement at, in, on, or near bone and/or chondral defect sites.

Example 14

Formation of Shapes or Sheets from Insoluble and Soluble Products

The dry soluble portion from Example 5 is dissolved in a 4.75% v/vethanol solution in water. Specifically, 1.2 g of the soluble portionwas dissolved in 40 mL of 4.75% v/v ethanol. Once a homogeneous solutionis obtained, 2.3 g of insoluble particles from example 6 are added. Thedispersion is thoroughly mixed and cast as a sheet. The mix isimmediately frozen and then lyophilized. Cancellous bone and/or DBM mayalso be added alone as insoluble particles or in combination with oneanother. This material may be used for placement at, in, on, or nearbone and/or chondral defects.

Example 15

Use of an Extrudable Product as Osteogenic Coating

The DBM derived product from Examples 1–14 is used to coat a surgicalbone implant. The coated implant is lyophilized. The lyophilized productmay be inserted at, in, on, or near a bone and/or chondral defect site.

Example 16

Formulation of an Extrudable Product Containing Cancellous Bone with orwithout the Addition of Blood or Bone Marrow

The DBM-derived gel and putty products from Examples 1–15 are combinedwith cancellous bone material to produce a gel or putty with differenthandling characteristics. In addition, the cancellous containinggel/putty can be combined with blood and/or bone marrow to provide agrowth factor-enriched osteogenic material.

Example 17

Illustrations of Bone Inductive Properties

Samples from Example 1 (25 mg lyophilized soluble material alone),Example 2 (25 mg lyophilized soluble material alone), Example 3 (25 mgrehydrated residual), Example 8 (25 mg of product as described inExample 8 plus DBM and residual), and Example 10 (25 mg of product asdescribed in Example 10) were prepared for implantation. These samples,along with samples of active DBM used to create the samples, weresterilized using electron-beam technology and implanted into themusculature of athymic (rhu/rhu) mice, according to IACUC approvedprotocols. The mice were anesthetized using a ketamine/xylazine mixturefor induction and maintenance of anesthesia throughout the procedure.General anesthesia was accomplished in approximately 3–5 minutes and wasverified by a lack of response to a toe pinch.

The dorsal area of each mouse was swabbed using betadine/alcohol scrub.Each mouse was placed in dorsal recumbency. Then using a scalpel orscissors, a 1 cm incision was made in the skin. A 1.0 mm incision wasmade in the muscle and blunt dissection was used to expand theimplantation site. All implants were placed between the muscle bundles.The procedure was repeated on the contralateral side through the sameskin incision. Wound closure was accomplished with one or two vicrylsutures for muscle closure and using stainless steel wound clips for theskin incision. Post-operative checks were made to ensure animals fullyrecover from the procedure.

On day 28 post-operatively, all animals were anesthetized with sodiumpentobarbital and sacrificed by cervical dislocation. After euthanasia,the skin over the implant was reflected and both implants were removed.The right implant from each animal was placed in 10% neutral bufferedformalin for histology and the left implant from each animal was placedin cryovials then flash frozen in liquid nitrogen for alkalinephosphatase analysis. Frozen specimens were stored at −80° C. forsubsequent biochemical analysis. The remaining specimens were placed in10% v/v neutral buffered formalin prior to histological processing.These samples were processed and embedded in paraffin. Decalcifiedparaffin embedded histological sections were prepared and stained withhaematoxylin and eosin. Qualitative analysis was performed to evaluateeach sample for bone inductivity. Samples from each paraffin embeddedsection were also evaluated quantitatively to determine the amount ofnewly formed bone as a function of total implant area.

The results from this study show that 25 mg (containing a little over 3times the amount of soluble portion present in the putty formulations)of 2.0 M—2 day lyophilized soluble material (soluble portion) alone isnon-inductive in this model. Conversely, 25 mg (a little over 3 timesthe amount of soluble portion present in the putty formulations) of 3.0M—2 day lyophilized dry material (soluble portion) is inductive suchthat very small ossicles formed in 5 of the 6 samples evaluated. The 2.0M—2 day plus DBM putty (FIGS. 1A and 1B) and the 3.0 M—2 day plusresidual DBM putty (FIGS. 2A and 2B) were also inductive in this model.

Example 18

Preparation of Bone Repair Composition

This process is conducted primarily at room temperature in amanufacturing environment with a controlled temperature of about 59–70°F. (15–21° C.). Certain steps are conducted well below room temperature,including freezing and lyopholization (freeze-drying) steps.

Demineralized bone matrix (DBM) particles are obtained from an AATBaccredited or other tissue bank or prepared by demineralizingmineralized bone by conventional methods. One lot of DBM particles isdivided into a first portion (approximately 60% of the lot) and a secondportion (approximately 40% of the lot). The DBM particles are thenplaced in an Erlenmeyer flask outfitted with a perforated Teflon™baffle. A 3 M citric acid solution is added (20 ml of acid per gram ofDBM particles). The flask stopper is outfitted with a fixture forattachment of a vacuum line connected to a pump supplying about 28 mm Hgvacuum. The assembly is affixed to an orbital shaker providing vigorousagitation. Vacuum and agitation are applied for 72 hours. Alternatively,this process can be conducted without application of a vacuum. Duringthis time, the temperature of the flask contents are at or slightlybelow room temperature. After 72 hours, the flask contents consist oftwo portions: an acidic liquid containing the soluble part of the DBMparticles and an insoluble solid particulate.

The entire contents of the flask is vacuum filtered through a 350 micronscreen on a Buchner funnel to separate the insoluble solid particulatefrom the acidic liquid containing the soluble part of the DBM particles.Both parts are retained.

The insoluble solid particulate is rinsed multiple times with roomtemperature deionized water in an ultrasonic cleaner. The washedparticles are drained, placed in sealed Tyvek™ bags, or other suitablecontainer, deep frozen in a freezer maintained at about −75 to −80° C.,and then lyophilized for 5–7 days. After lyophilization, the coolingsource is automatically turned off and the particles return to roomtemperature.

The acidic liquid portion obtained from the filtering step is dialyzedfor 72 hours in room temperature deionized water. The dialyzed materialis then placed in sealed containers, deep frozen in a freezer maintainedat −75 to −80° C., then lyophilized over several days to remove water.

Two separate and distinct components result from these processing steps:a light, dry, essentially white, fluffy material from the solubilizedportion of the DBM particles and a yellowish, dry particulate from theinsoluble solid particles.

Then, the two components are combined at room temperature. First, thefluffy component (dried soluble extraction product) is placed in adisinfected plastic container and partially dissolved in roomtemperature distilled water. The pH is then adjusted to about 1.8–2.2over several minutes by dropwise addition of room temperature 3 M citricacid. Then, the insoluble solid particles and the second portion of theDBM particles are added and mixed.

This final formulation is introduced into syringes and chilled atrefrigerator temperature (about −4° C.) for 8–72 hours.

The raw materials, intermediate materials or final product may besterilized, for example, by electron beam (“E-beam”) sterilizationtreatment, preferable at a target dose within 5–25 kilograys (kGy).

Example 19

Preparation of Bone Repair Composition in the Form of a Sponge

This process is conducted primarily at room temperature in amanufacturing environment with a controlled temperature of about 59–70°F. (15–21° C.). Certain steps are conducted well below room temperature,including freezing and lyopholization (freeze-drying) steps.

The dried soluble extraction product as prepared in Example 18 is usedfor this process. 2.5137 grams of DBM particles and 826.87 milligrams ofthe white, fluffy dried soluble extraction product are weighed. Thewhite, fluffy material is chopped into small pieces of about 1 to about2 cm. The chopped material is placed into a centrifuge tube(approximately 50 ml tube) or other similarly sized container. About 35ml of refrigerated 4.75% ethanol is added to the centrifuge tube and thecontents are aggressively mixed, for example, with a vortex mixer forapproximately 3 minutes or until the white, fluffy material isdispersed. The DBM particles are then added to the centrifuge tube andthe contents are aggressively mix for another 1 minute or until thecontents are thoroughly and uniformly mixed. Ethanol is then added tothe tube such that the contents fill the tube, for example, about 40 ml.The contents of the tube is again aggressively mixed for about 15seconds or until thoroughly mixed.

The mixture is then poured into a tray having the shape of the desiredproduct. The tray is placed in a refrigerator (˜4° C.) for about an houror until a firm gel is obtained. The tray is then transferred to afreezer of about −84° C. for at least 3 hours. The composition is thenlyophilized for about 24 hours, or until the composition issubstantially free of moisture. The end product has a sponge-likeconsistency. The thickness may vary, but preferably is ⅛″ to about 1″thick, more preferably about ¼″ thick.

1. An osteogenic composition resulting from a process comprising: (a)subjecting demineralized bone to an acidic extraction medium at a pH ofbetween about 0.5 and about 5.5 at an extraction temperature betweenabout 15° C. and 25° C. to produce an insoluble extraction product and asoluble extraction product; (b) separating the insoluble extractionproduct and the soluble extraction product; (c) drying the solubleextraction product to remove all or substantially all of the moisture inthe soluble extraction product to produce a dried soluble extractionproduct; and (d) combining the dried soluble extraction product of step(c) with demineralized bone particles.
 2. The osteogenic composition ofclaim 1, wherein the dried soluble extraction product is subsequentlyhydrated and the composition has the consistency of a gel, paste orputty.
 3. The osteogenic composition of claim 1, wherein the processfurther comprises the step of (e) combining the dried soluble extractionproduct with an alcohol and removing the alcohol by drying, and whereinthe composition has the consistency of a sponge.
 4. The osteogeniccomposition of claim 1, wherein step (a) is conducted under vacuum. 5.The composition of claim 1, wherein the soluble extraction product isosteoinductive.
 6. The composition of claim 1, wherein the extractionmedium comprises citric acid, hydrochloric acid, acetic acid, lacticacid or malic acid.
 7. The composition of claim 1, further comprisingsterilizing the osteogenic composition.
 8. The composition of claim 7,wherein the sterilizing is conducted by electron beam radiationsterilization.
 9. The composition of claim 1, wherein the drying of step(c) comprises lyophilization.
 10. The composition of claim 1, whichfurther comprises the insoluble extraction product.
 11. A method forpreparing an osteogenic composition comprising: (a) subjectingdemineralized bone to an acidic extraction medium at a pH of betweenabout 0.5 and about 5.5 at an extraction temperature between about 15°C. and 25 ° C. to produce an insoluble extraction product and a solubleextraction product; (b) separating the insoluble extraction product andthe soluble extraction product; (c) drying the soluble extractionproduct to remove all or substantially all of the moisture in thesoluble extraction product to produce a dried soluble extractionproduct; and (d) combining the dried soluble extraction product of step(c) with demineralized bone particles.
 12. The method of claim 11,wherein the dried soluble extraction product is subsequently hydratedand the composition has the consistency of a gel, paste or putty. 13.The method of claim 11, wherein the method further comprises the step ofcombining the dried soluble extraction product with an alcohol andremoving the alcohol by drying, and wherein the composition has theconsistency of a sponge.
 14. The method of claim 11, wherein step (a) isconducted under vacuum.
 15. The method of claim 11, wherein the solubleextraction product is osteoinductive.
 16. The method of claim 15,wherein the extraction medium comprises citric acid, hydrochloride acid,acetic acid, lactic acid or malic acid.
 17. The method of claim 11,further comprising sterilizing the osteogenic composition.
 18. Themethod of claim 17, wherein the sterilization is conducted by electronbeam radiation sterilization.
 19. The method of claim 11, wherein thedrying of step (c) comprises lyophilization.
 20. The method of claim 11,wherein the composition further comprises the insoluble extractionproduct.
 21. An osteogenic surgical implant comprising a surgicalimplant coated with the composition of claim
 1. 22. A method of treatinga bone defect comprising: (a) providing a surgical implant; (b) coatingthe surgical implant with the composition of claim 1; and (c) implantingthe surgical implant at a bone defect site.
 23. A composition comprisinga separated soluble extraction product derived from demineralized bonecontacted with acidic extraction medium at a pH of between about 0.5 andabout 5.5 at an extraction temperature between about 15° C. and 25° C.24. The composition defined in claim 23 wherein the product furthercontains proteins endogenous to the demineralized bone.
 25. Thecomposition defined in claim 24 further comprising an insolubleextraction product derived from demineralized bone.
 26. The compositiondefined in claim 24 further comprising demineralized bone.
 27. Thecomposition defined in claim 26 further comprising an insolubleextraction product from demineralized bone.
 28. The composition definedin claim 27 wherein the composition comprises only materials derivedfrom bone.
 29. The composition defined in claim 27 wherein thecomposition consists essentially of the soluble extraction product, theinsoluble extraction product and the demineralized bone.
 30. Thecomposition defined in claim 23 further comprising at least onecomponent selected from the group consisting of bone morphogeneticproteins, cartilage derived morphogenetic proteins (CDMPs), corticalbone, cancellous bone, bone mineral, osteogenic chemical, osteogenicpeptide, osteogenic growth factor, recombinant bone morphogeneticprotein, fibronectin, blood-derived protein, thickening material,collagen, insoluble extraction product, calcium phosphate,hydroxyapatite, tricalcium phosphate, biphasic calcium phosphate,calcium sulfate, biological glass, natural polymer, synthetic polymer,poloxamer, and reverse phase medium.
 31. The composition defined inclaim 30 wherein the component is cancellous bone.
 32. The compositiondefined in claim 30 wherein the component is bone mineral.
 33. Thecomposition defined in claim 30 wherein the component is at least onecomponent selected from the group consisting of hydroxyapatite,tricalcium phosphate, biphasic calcium phosphate, and poloxamer.
 34. Thecomposition defined in claim 30 wherein the component is poloxamer. 35.The composition defined in claim 33 further comprising demineralizedbone.
 36. The composition defined in claim 34 further comprisingdemineralized bone.
 37. The composition defined in claim 34 furthercomprising an insoluble extraction product from demineralized bone. 38.The composition defined in claim 36 further comprising an insolubleextraction product from demineralized bone.
 39. A method of making anosteogenic material comprising: (a) subjecting demineralized bone matrixto an acidic extraction medium at a pH of between about 0.5 and about5.5 at an extraction temperature between about 15° C. and 25° C. underconditions to produce an insoluble extraction product and a solubleextraction product; and (b) separating the insoluble extraction productand the soluble extraction product.
 40. The method defined in claim 39further comprising (c) treating the soluble extraction product to removeall or substantially all of the moisture in the soluble extractionproduct.
 41. The method defined in claim 40 further comprising (d)combining the treated soluble extraction product of step (c) withdemineralized bone.
 42. The method defined in claim 41 furthercomprising (e) combining the treated combination of step (d) with theinsoluble extraction product.
 43. The method defined in claim 40 furthercomprising adding the treated soluble extraction product of step (c) toan alcohol; and removing the alcohol; wherein the composition has theconsistency of a sponge.
 44. The method defined in claim 40 furthercomprising hydrating the treated soluble extraction product of step (c);wherein the hydrated composition has the consistency of a gel, paste orputty.
 45. The method of claim 40, wherein step (a) is conducted undervacuum.
 46. The method of claim 39, wherein the soluble extractionproduct is osteoinductive.
 47. The method of claim 46, wherein theextraction medium comprises citric acid, hydrochloric acid, acetic acid,lactic acid or malic acid.
 48. The method of claim 40, furthercomprising sterilizing the treated soluble extraction product.
 49. Themethod of claim 41, further comprising sterilizing the composition ofstep (d).
 50. The method of claim 42, further comprising sterilizing thecomposition of step (e).
 51. The method of claim 40, wherein thetreating of step (c) comprises lyophilization.
 52. An osteogenicsurgical implant comprising a surgical implant coated with thecomposition of claim
 23. 53. A method of treating a bone defectcomprising applying to a bone defect an effective amount of thecomposition defined in claim
 25. 54. A method of treating a bone defectcomprising applying to a bone defect an effective amount of thecomposition defined in claim
 26. 55. An osteogenic composition resultingfrom a process comprising: (a) subjecting demineralized bone to anacidic extraction medium at a pH of between about 0.5 and about 5.5 atan extraction temperature of about room temperature to produce aninsoluble extraction product and a soluble extraction product; (b)separating the insoluble extraction product and the soluble extractionproduct; (c) drying the soluble extraction product to remove all orsubstantially all of the moisture in the soluble extraction product toproduce a dried soluble extraction product; and (d) combining the driedsoluble extraction product of step (c) with demineralized boneparticles.
 56. A method for preparing an osteogenic compositioncomprising: (a) subjecting demineralized bone to an acidic extractionmedium at a pH of between about 0.5 and about 5.5 at an extractiontemperature of about room temperature to produce an insoluble extractionproduct and a soluble extraction product; (b) separating the insolubleextraction product and the soluble extraction product; (c) drying thesoluble extraction product to remove all or substantially all of themoisture in the soluble extraction product to produce a dried solubleextraction product; and (d) combining the dried soluble extractionproduct of step (c) with demineralized bone particles.
 57. A compositioncomprising a separated soluble extraction product derived fromdemineralized bone contacted with acidic extraction medium at a pH ofbetween about 0.5 and about 5.5 at an extraction temperature of aboutroom temperature.
 58. The composition of claim 1, wherein the extractionmedium comprises 1.0M to 10.0 M citric acid.
 59. The composition ofclaim 1, wherein the extraction medium comprises 2.0M to 3.0 M citricacid.
 60. The composition of claim 1, wherein the extraction mediumcomprises 3.0M citric acid.
 61. The composition of claim 11, wherein theextraction medium comprises 1.0M to 10.0 M citric acid.
 62. Thecomposition of claim 11, wherein the extraction medium comprises 2.0M to3.0M citric acid.
 63. The composition of claim 11, wherein theextraction medium comprises 3.0M citric acid.
 64. The composition ofclaim 23, wherein the soluble extraction product is osteoinductive. 65.The composition of claim 23, wherein the extraction medium comprisescitric acid, hydrochloric acid, acetic acid, lactic acid or malic acid.66. The composition of claim 23, wherein the extraction medium comprises1.0M to 10.0 M citric acid.
 67. The composition of claim 23, wherein theextraction medium comprises 2.0M to 3.0M citric acid.
 68. Thecomposition of claim 23, wherein the extraction medium comprises 3.0Mcitric acid.
 69. The composition of claim 39, wherein the extractionmedium comprises 1.0M to 10.0 M citric acid.
 70. The composition ofclaim 39, wherein the extraction medium comprises 2.0M to 3.0M citricacid.
 71. The composition of claim 39, wherein the extraction mediumcomprises 3.0M citric acid.
 72. The composition of claim 55, wherein thesoluble extraction product is osteoinductive.
 73. The composition ofclaim 55, wherein the extraction medium comprises citric acid,hydrochloric acid, acetic acid, lactic acid or malic acid.
 74. Thecomposition of claim 55, wherein the extraction medium comprises 1.0M to10.0 M citric acid.
 75. The composition of claim 55, wherein theextraction medium comprises 2.0M to 3.0M citric acid.
 76. Thecomposition of claim 55, wherein the extraction medium comprises 3.0Mcitric acid.
 77. The composition of claim 56, wherein the solubleextraction product is osteoinductive.
 78. The composition of claim 56,wherein the extraction medium comprises citric acid, hydrochloric acid,acetic acid, lactic acid or malic acid.
 79. The composition of claim 56,wherein the extraction medium comprises 1.0M to 10.0 M citric acid. 80.The composition of claim 56, wherein the extraction medium comprises2.0M to 3.0M citric acid.
 81. The composition of claim 56, wherein theextraction medium comprises 3.0M citric acid.
 82. The composition ofclaim 57, wherein the soluble extraction product is osteoinductive. 83.The composition of claim 57, wherein the extraction medium comprisescitric acid, hydrochloric acid, acetic acid, lactic acid or malic acid.84. The composition of claim 57, wherein the extraction medium comprises1.0M to 10.0 M citric acid.
 85. The composition of claim 57, wherein theextraction medium comprises 2.0M to 3.0M citric acid.
 86. Thecomposition of claim 57, wherein the extraction medium comprises 3.0Mcitric acid.